1. Field of the Invention
This invention relates to an activated matrix and to a method of activating the matrix. It further relates to the activated matrix further coupled to a ligand or leash and to a method of coupling the ligand or leash.
For the purposes of this specification, activating a matrix means carbonylating a matrix as defined in the appended claims with a suitable carbonylating agent. Activated matrix means a product of such a carbonylating reaction. Coupling a ligand or leash means substituting a ligand or leash having a good nucleophilic group, such as an amino group, into the carbonyl group of the activated matrix. A ligand is a substituent selected to be specifically attractive to a compound sought to be bound to the coupled matrix. Such binding may be by ion exchange or affinity chromatography, or for a radioimmuno assay, for hydrophobic chromatography or as an enzyme support or the like. A leash is a substituent which can be readily substituted into the carbonyl group of the activated matrix and then itself be substituted by a ligand. A macroporous synthetic polymer means a synthetic polymer which is sufficient porous to allow adsorbtion of macromolecules within the polymer. Coated glass beads refer to silica coated with a hydroxyl containing organic group such as an alkyl group by using a reactive silylating reagent which contains the necessary organic group (or precursor) attached to it by a stable Si-C bond. The hydroxyl groups on (or subsequently formed on) the organic group react with the carbonylating reagent to form an activated matrix.
The expression "a rigid support" is used in respect of a substance such as silica to which can be bonded directly an hydroxyl containing organic group as stated above.
2. Description of the Prior Art
Affinity chromatography is a separation technique exploiting the unique specificity of biological interaction to isolate naturally occurring compounds such as proteins, polysaccharides, glycoproteins and nucleic acids. Specific adsorptive properties are imparted to a bed material by covalently bonding an appropriate binding ligand to an insoluble matrix, i.e. by attachment of a suitable protein. The ligand acts to adsorb from solution a substance to be isolated, the isolated substance subsequently being dissolved by changing the experimental conditions after unbound substances have been washed away. The high specificities exploited by this method are natural specificities such as, for example, antigen/antibody, enzyme/inhibitor or hormone/carrier. Isolation of substances through this technique differs from conventional chromatography in which separation depends on gross physical and chemical differences between the substances.
Affinity chromatographic matrices are prepared by first activating a polysaccharide and then coupling a ligand and leash when required. In one such preparation agarose is reacted in a two-stage process as set out below. The first stage comprises treatment with cyanogen bromide to form an imido-carbonate compound of Formula I and the second stage comprises treatment with a primary amine to form an isourea compound of Formula II as set out hereinbelow: ##STR1## wherein R is a suitable affinity chromatography ligand. In the second stage reaction involving a nitrogen nucleophile the matrix becomes, in part, an ion exchange resin charged with the basic isourea group. This charge is present at pH 7 thus interfering with the specificity of the compound when attempting to isolate substances from biological materials. It also remains, but to a lesser extent, a higher pH levels. It will be appreciated that the specificity of the ligand would be considerably reduced by the presence of the charged groups thus reducing the desirability of the matrix activated by this method.